5,6-trans-25-hydroxycholecalciferol

ABSTRACT

5,6-TRANS-25-HYDROXYCHOLECALCIFEROL. THE COMPOUND EXHIBITS SOME ANTIRACHITIC ACTIVITY BUT IS PRIMARILY CHARACTERIZED BY ITS ABILITY TO STIMULATE INTESTINAL CALCIUM TRANSPORT WHILE HAVING LITTLE IF ANY ABILITY TO STIMULATE MOBILIZATION OF CALCIUM FROM BONE IN NEPHRECTOMIZED ANIMALS.

United States Patent 3,743,661 5,G-TRANS-ZS-HYDROXYCHOLECALCIFEROL Hector F. De Luca, Michael F. Holick, and Michele Garabedian, Madison, Wis., assiguors to Wisconsin Alumni Research Foundation, Madison, Wis. No Drawing. Filed Feb. 14, 1972, Ser. No. 226,183

Int. Cl. C07c 171/10 US. Cl. 260397.2 1 Claim ABSTRACT OF THE DISCLOSURE 5,6-trans-25-hydroxycholecalciferol. The compound exhibits some antirachitic activity but is primarily characterized by its ability to stimulate intestinal calcium transport while having little if any ability to stimulate mobilization of calcium from bone in nephrectomized animals.

The invention described herein was made in the course of work under a grant or award from the Department of Health, Education, and Welfare.

This invention relates to a derivative of vitamin D More specifically this invention relates to an isomer of 25-hydroxycholecalciferol.

The character and activity of vitamin D and vitamin D are well documented and the 5,6-trans isomers of these vitamins have been identified, isolated and synthesized (see for example A. Verloop et al., Rec. Trav. Chim., 74, 1125 (1955) and H. H. Inhofien et al., Chim. Ber., 90, 2544 (1957)).

In recent years derivatives of vitamins D and D which are more active than the 'D-vitamins themselves have been isolated, identified and synthesized. Among these is 25-hydroxycholecalciferol which exhibits greater antirachitic activity than vitamin D (See U.S. Letters Patent No. 3,565,924, issued Feb. 23, 1971.)

The 5,6-trans isomer of 25-hydroxycholecalciferol has now been prepared and has been found to exhibit some unexpected biological properties as will be more apparent from the following description. Specifically, this compound is 9,10-seco (5E,7E) 5,7,10(19)-cholestatriene- 3 3,25-diol but will be referred to hereinafter as 5,6-trans- 25-HCC.

5,6-trans-25-HCC was prepared as follows:

Ten mg. of 25-hydroxycholecalciferol (25 HCC) was dissolved in ml. in a 9:1 admixture of Skellysolve B (a light petroleum fraction redistilled at 67-69 C.) and diethylether (v./v.). 50 ,ul. of a solution I (0.1 mg./1 ml. Skellysolve B) was subsequently added. After 1 hr. at 25 C. the reaction was terminated wtih solid Na S O washed with water and the product in the Skellysolve B/ ether phase was dried over anhydrous Na SO.,. The solvent was evaporated under N and the product was redissolved in 1 ml. of a 7:3 mixture Skellysolve B/diethylether (v./v.). The sample was applied to a g. multibore silicic acid column measuring stepwise in diameter 1.2, 0.8 and 0.4 cm. The column was eluted with a hyperbolic gradient generated by having 230 ml. [7:3 Skellysolve B/diethylether (v./v.)] in a mixing chamber and 400 ml. [3:7 Skellysolve B/diethylether (v./v.)] in a holding chamber. Three-hundred ml. of diethylether was added to the holding chamber after it emptied. One-hundred 5 ml. fractions were collected and the ultraviolet absorption spectra of each was taken to determine the elution position of the 5,6-trans-25-HCC.

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The isomer was collected and chromatographed once again on the multibore silicic acid column.

The 25-hydroxycholecalciferol starting material in the above synthesis can be prepared as described by I. W. Blunt H. F. DeLuca in Biochemistry '8, p. 671 (1969).

Structural identification of 5,6-trans-25-OH- cholecalciferol The ultraviolet spectrum of the isomer showed the characteristic A 273.5 nm. and a A 232 nm. (6,7) for the 5,6-trans-triene system. The mass spectrum of the analog showed a molecular ion at m/e 400 similar to 25-HCC and fragments at m/e 271 and 253 (271-H O) characteristic for loss of side chain and at m/e 136 and 118 (136-H O) characteristic for the A ring plus C and C Gas-liquid chromatography (GLC) of the 5,6-trans- 25-HCC showed only one component which is consistent with a previous report for 5,6-trans-vitamin D, (A. Verloop et al., Rec. Trav. Chim., 78, 1004 (1959)). The GLC chromatogram also demonstrates the absence of 25-HCC which normally has a shorter retention time and appears as two peaks due to thermal cyclization to the pyro and isopyro forms of the vitamin.

BIOLOGICAL ACTIVITY Antirachitic activity Antirachitic activity of 5,6-trans-25-HCC was measured by the standard line test assay as described in US. Pharmacopoeia, 15th Revision (Mack Publishing Co., Easton, Pa. 1955), p. 889. The following results, which represent the average values from eight animals in each case, were obtained.

Table 1 Compound: Antirachitic activity (IU/ g.) Vitamin D 45:5 5,6-trans-25-HCC 4i 1 Intestinal calcium transport For intestinal calcium transport measurements, weanling male albino rats, obtained from the Holtzman Co., Madison, Wis., were housed individually in hanging wire cages and given food and water ad libitum. They were fed for three weeks the low calcium purified diet described by Suda et al. (J. Nutr. 100, 1049 (1970)). The rats became severely vitamin D-deficient as revealed by a serum calcium concentration of 4-5 mg./ 100 ml. At this time the rats were divided into five groups. One group received only the ethanol vehicle. Two other groups (either sham operated, i.e. incised to induce like trauma but without kidney removal or bilaterally nephrectomized) received either 25 ,ug. of 5,6-trans-25- HCC or 25 ,ug. of 25-HCC dissolved in the ethanol. All doses were administered intrajugularly in 0.05 ml. EtOH. Sixteen hours later the rats were decapitated. The small intestines were removed for the measurement of intestinal calcium transport by the everted gut sac technique described by Martin and DeLuca (Am. J. 'Physiology 216, 1351 (1969). The proximal 5.5 cm. of the duodenum was used in the test.

The results obtained are shown in the table below.

TABLE 2 TABLE 3 45 Ca Serosall Dose Serum Ca++ Dose Condition of animal 45 Ca Mucosal Control 4.3=l:0.1(6) Control Normal 1. 8;b0.2(5) 25 #g. 5,6-trans-25HOC Bilaterally nephreetomized.. 4. 9:1:0.1(6) 25 g. 5,6-trans C Sham operated 4t t:|=0.3 0.25 g. 25-HOC .do 4. 55:0. 1(6) Do Bilaterally nephreeto- 3. 8=|;0.3(6)

Inized. 1 Plus or minus the standard error of the mean. The numbers in paren- 25 gjg5-HOQ Shad; 0 e ateh theses show the number of rats in each group.

I l p Table 3 clearly demonstrates that the 5,6-trans-25- gg figif fg gg a ffiggfgg fggg gggg Numbers parentheses 10 HCC lilce 2 5-HCC itself has little or no activity in elicit- Percent. mg a rise in serum calcium at the expense of bone 1n Calcium mobilization from bone nephrectomized rats- H Although it is evident from the foregoing data that For the measurement of calcium mobilization from 5 5 is only about one tenth as active as bone weanhng Holtzman P w fed P q vitamin D in the cure of rickets it exhibits the unusual quate calcium and phosphorus, vitamin D-deficient diet operties of being able to stimulate intestinal calcium descrlbed 111 y yi 204, P- 83816111011. et transport in bilaterally nephrectomized rats while having for two then the 1ow 0110111111 little or no activity in stimulating the mobilization of Vltamm D'defiQent dlet of Suda et p for calcium from bone. This suggests the application of 5,6- othqr 10 y r up m e bllaterally nephrectrans-ZS-HCC in the treatment of bone disease assotomized and were in ected immediately after surgery with ciated with chromic renal failure elther Having thus described the invention what is claimed is: solved in ,ul. of ethanol. Controls received 50 5 6 t 25 h d h l l if l ,ul. of 95% ethanol vehicle. Twenty-four hours after the administration of the dose, the animals were killed by References Cited decapitation and the blood serum was collected. 25

Serum calcium determinations were made with an UNITED STATES PATENTS atomic absorption spectrophotometer (Perkin-Elmer 3,585,221 6/1971 DeLuca et a1 260-3972 Model #214). For this, serum samples (0.10 ml.) were 3,565,924 2/1971 DeLuca et a1 diluted with 1.9 ml. of 0.1% LaCl t Results are shown below 30 ELBERT L. ROBERTS, Primary Examiner 

